Biosensing systems for measurement of lactose

• US 10,024,797 B2
• Filed: 11/22/2011
• Issued: 07/17/2018
• Est. Priority Date: 11/22/2010
• Status: Active Grant

• Current Assignee: Colorado State University Research Foundation (Colorado State University System)
• Original Assignee: Colorado State University Research Foundation (Colorado State University System)
• Inventors: Reardon, Kenneth F., Dandy, David S., Holcomb, Ryan E.
• Primary Examiner(s): Tate, Christopher R
• Assistant Examiner(s): Kosar, Aaron J
• Application Number:
  US13/988,723
• Publication Number:
  US 20130244266A1
• Time in Patent Office: 2,429 Days
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: G01N 2021/772 Tip coated light guide
• CPC Class Codes: G01N 2021/773 Porous polymer jacket; Polymer matrix with indicator
• CPC Class Codes: G01N 2021/775 Indicator and selective membrane
• CPC Class Codes: G01N 2021/7786 Fluorescence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 21/7703 using reagent-clad optical fibres or optical waveguides using measurement of total internal reflection or attenuated total reflection G01N21/552; optical fibres or waveguides per se G02B

Abstract

Herein disclosed are biosensing systems that measure lactose concentration in a solution.

Claims

1. A biosensing system that measures lactose concentration in a solution, said biosensing system comprising:

an optode comprising an optical fiber having a first tip and a second tip,

a luminescent transducer layer covering said first tip,

a biocomponent layer covering said luminescent transducer layer,

said biocomponent layer comprising at least one biocomponent that catalyzes a reaction with lactose in said solution, wherein

said at least one biocomponent is immobilized within said biocomponent layer using a hydrogel or polymer matrix,

a porous membrane covering said biocomponent layer, wherein said porous membrane provides a mass transfer resistance between said biocomponent layer and said solution,

a photon-detection device coupled to said second tip, wherein the photon-detection device is configured to detect photons emitted from the luminescent transducer layer through the optical fiber, and

a signal processing system coupled to said photon-detection device.

2. The biosensing system of claim 1 whereby said lactose concentration in said solution, the depth of said biocomponent layer, the depth of said porous membrane, the diffusion coefficient of lactose in said porous membrane, and the K_M and V_max of said catalytic reaction between said biocomponent and lactose are selected such that Da is greater than the value of 1−

β and

the quotient between Da² and 4β

is from about 10 to at least 1000,

wherein V_max is the maximum reaction rate of said biocomponent with lactose under saturating lactose concentrations, and

wherein K_M is the lactose concentration at which the reaction rate of said biocomponent and lactose is half of V_max, and

wherein β

is the lactose concentration in said solution divided by said K_M of said biocomponent for lactose, and

wherein Da is a dimensionless Damkö

hler number, and

wherein Da is (h_eV_maxh_p)/(D_pK_M), and

wherein h_e is the thickness of said biocomponent layer, and

wherein h_p is the thickness of said porous membrane, and

wherein D_p is the diffusion coefficient of lactose in said porous membrane.

3. The biosensing system of claim 1 wherein said luminescent transducer layer contains a luminescent agent that is selected from the group consisting of

a fluorescent agent,

a phosphorescent agent,

a bioluminescent agent, or

a chemiluminescent agent.

4. The biosensing system of claim 1 wherein said luminescent transducer layer contains a luminescent agent that is selected from the group consisting of:

trisodium 8-hydroxy-1,3,6-trisulphonate, fluoro (8-anilino-l-naphthalene sulphonate), tris(bipyridine)ruthenium(II) complex, Tris(4,7-diphenyl-1,10-phenanthroline)Ru(II) (RuDPP), ruthenium complexes, and acridinium- and quinidinium-based reagents, fluorescein, fluoresceinamine, or a fluorescein containing compound.

5. The biosensing system of claim 1 wherein said biocomponent layer comprises a biocomponent selected from the group consisting of at least one enzyme from Enzyme Commission numbers 1.1.3, 1.2.3, 1.3.3, 1.4.3, 1.5.3, 1.6.3, 1.7.3, 1.8.3, 1.9.3, 1.10.3, 1.16.3, 1.17.3, 1.21.3, and 3.2.1.23.

6. The biosensing system of claim 1 wherein said polymer matrix comprises one or more of polyacrylamide, polystyrene, polymethacrylate, polyvinylalcohol and polyurethane, and wherein said biocomponent is entrapped within said polymer matrix.

7. The biosensing system of claim 6 wherein said biocomponent is bound to said matrix layer through adding crosslinking agents to said biocomponent entrapped within said matrix layer, and

wherein said crosslinking agents are selected from the group consisting of glutaraldehyde, hexamethylene diisocyanate and 1,5-dinitro-2,4-difluorobenzene, polyethyleneimine, hexamethylenediamine and formaldehyde.

8. The biosensing system of claim 1 wherein said luminescent transducer layer is bound in a layer of molecules bound to the first tip of said optical fiber, said layer of molecules is selected from the group consisting of cellulose, cellulose derivatives, silica, glass, dextran, starch, agarose, porous silica, chitin and chitosan.

9. The biosensing system of claim 1, said porous membrane is polycarbonate having a pore size of from about 0.005 μ

m to about 0.025 μ

m.

10. The biosensing system of claim 9, said porous membrane comprises a coating of polyurethane.

11. The biosensing system of claim 1 wherein said biocomponent comprises beta-galactosidase and glucose oxidase and wherein luminescence of said luminescent transducer layer is at least partially quenched with oxygen.

12. The biosensing system of claim 11 wherein said biocomponent further comprises catalase.

13. The biosensing system of claim 1 wherein said biocomponent comprises beta-galactosidase and galactose oxidase and wherein luminescence of said luminescent transducer layer is at least partially quenched with oxygen.

14. The biosensing system of claim 13 wherein said biocomponent further comprises catalase.

15. The biosensing system of claim 1 wherein said biocomponent is carbohydrate oxidase and wherein luminescence of said luminescent transducer layer is at least partially quenched with oxygen.

16. The biosensing system of claim 1 wherein said biocomponent further comprises catalase.

17. The biosensing system of claim 1 wherein said biocomponent is cellobiose dehydrogenase and wherein luminescence of said luminescent transducer layer interacts with protons.

18. The biosensing system of claim 1 wherein said biocomponent comprises beta-galactosidase and hexose oxidase and wherein luminescence of said luminescent transducer layer is at least partially quenched with oxygen.

19. The biosensing system of claim 18 wherein said biocomponent further comprises catalase.

20. The biosensing system of claim 1, wherein said hydrogel comprises one or more of algal polysaccharides, agarose, alginate, gelatin, collagen, pectin, poly(carbamoyl)sulfonate, locust bean gum, and gellan, and wherein said biocomponent is entrapped within said hydrogel.

21. The biosensing system of claim 1, wherein said biocomponent layer further comprises one or more of a gel-hardening agent and a stabilizing agent.

22. The biosensing system of claim 21, wherein said one or more of a gel-hardening agent and a stabilizing agent comprises one or more of glutaraldehyde, polyethyleneimine, hexamethylenediamine and formaldehyde.

23. The biosensing system of claim 1, wherein said porous membrane enables said biosensing system to measure lactose in said solution at concentrations up to and including 20 mM.

24. The biosensing system of claim 1, wherein said porous membrane enables said biosensing system to measure lactose in said solution at concentrations up to and including 100 mM.

25. The biosensing system of claim 1, wherein said luminescent transducer layer comprises one or more fluorophores capable of interacting with protons.